1. Field of the Invention
This invention generally relates to methods for rapid and inexpensive determination of the level or concentration of total coliforms or E. coli in a liquid sample. The invention provides a simple assay using a reagent mixture containing an indicator compound which changes color upon cleavage by the beta galactosidase enzyme specifically found in coliforms or the beta glucuronidase enzyme unique to E. coli. These assays are particularly useful in detecting total coliform or E. coli contamination in recreational water, drinking water, food processing operations and other matrices which may be contaminated with coliforms and E. coli.
2. Description of the Prior Art
Pathogenic microorganisms, including coliforms, can be present in high concentrations in the feces of infected humans and animals. Contamination of water with sewage or fecal wastes may be a serious threat to human health. Contamination of meat and poultry by exposure to fecal material during processing also poses a threat to human health. The sanitary quality of drinking water, recreational water, wash water or areas in which butchering and processing take place must be carefully monitored.
Numerous methods for testing the sanitary quality of water are available but in general they require 24-48 h for growth of the microorganisms. These methods include monitoring for the presence of indicator bacteria, total coliforms and fecal coliforms. The total coliform group includes certain strains of bacteria which are generally not found in fecal material, i.e. Klebsiella, Enterobacter, Citrobacter, Serratia sp., as well as bacteria which may be found in soil and on vegetation but are infrequently isolated from feces. The fecal coliform group is comprised principally of the bacterium Escherichia coli, the predominant coliforms found in feces, but does include some Klebsiella and Enterobacter sp. E. coli are found in the intestines of humans and animals and are present at a cell density in the range of 10.sup.7 to 10.sup.8 cells per gram of feces.
Test methods for the determination of total coliforms include the use of chromogenic and fluorogenic chemicals, i.e. orthonitrophenyl galactoside (ONPG), chlorophenylred galactoside (CPRG), methylumbelliferone-galactoside, often coupled with membrane filtration technology. Test methods for detecting fecal coliforms also include but are not limited to the use of fluorogenic and chromogenic chemicals, i.e. methylumbelliferyl glucuronide (MUG), resorufin D glucuronide, and fluorescein diglucuronide. Techniques currently used require 24 to 48 hours to assess contamination levels and often require additional testing to confirm the presence of coliforms and/or E. coli.
Many commercially available assays for the detection of coliforms or E. coli use membrane filter methods from Standard Methods for Examination of Water and Waste Water (Standard Methods for Examination of Water and Waste Water, 18th ed., Greenberg et al. eds, pp. 9-45 to 9-64 1992!) in which a specific volume of water sample or dilution(s) of the water sample are passed through a sterile 0.45 .mu.m membrane filter. The filter is then removed from the filter apparatus and placed on top of a solid agar substrate for growth at a specified temperature for a specified time period. These assays require days for growth, enumeration and confirmation of the contaminant of interest. Other commercial assays use a reagent mixture to which the sample is added, or the sample to which the reagent is added (Colilert.RTM., Edberg U.S. Pat. Nos. 4,925,789 and 5,429,933; Colisure.TM., Millipore; Coliquik.TM., Hach), followed by incubation for 18-28 h to obtain presence/absence results with respect to coliform and E. coli contamination. Most Probable Number (MPN) analysis using the same or similar reagent mixtures can also be performed to yield the most probable number of coliforms and E. coli present in the water sample within 24-48 h.
U.S. Pat. No. 5,420,017 to Tuompo describes a method for detecting microorganisms by using a filter to trap microorganisms. A test solution is added which contains a chromogenic reagent which can be reduced by microbial dehydrogenase enzymes to yield a colored product. Detection limits range from 10.sup.5 to 10.sup.6 colony forming units (cfu)/ml.
U.S. Pat. No. 4,923,804 to Ley et al. describes a method that is specific for detecting and enumerating E. coli in a water sample. The method is carried out using standard membrane filtration analysis with a chromogenic reagent, which if subjected to .beta.-glucuronidase activity as a result of the presence of E. coli produces clearly defined coloring on the membrane filter representative of individual colonies grown from E. coli cells in the test specimen.
U.S. Pat. Nos. 4,925,789 and 5,429,933 to Edberg describe a method for determining the presence or absence of a predetermined target microbe in a sample by addition of test medium to the sample or of the sample to test medium. The test medium is a defined substrate providing a selective growth medium for the target microbe and includes a specific nutrient which only the target microbe can metabolize. The sample altering moiety attached to the specific nutrient is activated to change the sample only if the specific nutrient is metabolized by the target microbe. The sample-altering moiety can be a material which changes the color of the sample, an electrical characteristic of the sample or some other detectable characteristic of the sample. Sterility of the test matrix is not required. In addition, U.S. Pat. No. 5,429,933 includes the use of an antibiotic to inhibit growth of non-target microbes in the medium specified and an accelerant which hastens the growth of target microbes to the log phase of growth. U.S. Pat. No. 4,925,789 suggests that MPN analysis using the reagent mixture can be performed.
U.S. Pat. No. 5,610,029 to Ehrenfeld et al. describes an optimized medium for determining the presence of target microorganisms within 18-24 h. A "microbe-specific medium" allows substantial growth of only the target microbe and includes media containing one or more antibiotics specific for inhibiting growth of microorganisms other than the target microbe.
Detection of microbial by-products or metabolites as a means for detecting bacterial contamination have also been developed. Some of these techniques include detection of bacterial endotoxins using a Limulus Lysate Assay, detection of bacteria using electrical impedance measurements, ATP assays, and carbon-14 labeled substrate assays. These tests have not been widely accepted because they do not specifically determine the presence and/or concentration of microorganisms associated with human or animal intestinal flora which can be used to predict whether or not putative pathogens are present.